Quantitative Analysis of Salmon Calcitonin Hydroxyapatite Nanoparticle Permeation to substantiate Non-Invasive Bone Targeting via Sublingual Delivery

Abstract

We earlier reported comparable efficacy in bone parameters of sublingually administered salmon calcitonin hydroxyapatite nanoparticles (SCT-HAP-NPs) compared to the subcutaneous injection, in the ovariectomy rat model, despite a bioavailability of barely ~ 15%. We ascribed this intriguing finding to targeted bone delivery, facilitated by translocation of significant quantity of intact NP into systemic circulation. In the present study we track the translocation of FITC-SCT-HAP-NPs (~ 100 nm) across porcine sublingual mucosa using the Franz diffusion cell to validate our hypothesis. Confocal Laser Scanning microscopy (CLSM) established that SCT-HAP-NPs permeated into the deeper layers of sublingual porcine mucosal tissue. We confirmed the nanoparticles were present in the receptor medium of the Franz diffusion cell by DLS and TEM. We also demonstrate for the first time quantification of the NPs (%) translocated across the porcine mucosa, using the Amnis Image StreamX Mk II imaging flow cytometer. Computation revealed transport of ~ 60% of the FITC-SCT-HAP-NPs across mucosa in 2 h, substantiated that high NP concentrations could reach systemic circulation. Such high NP concentration in systemic circulation coupled with the small size (~ 100 nm) and the high bone affinity of HAP, validate our hypothesis of targeted bone delivery following sublingual administration. Furthermore, quantification of translocated NPs, which we demonstrate for the first time, would permit rational development of optimal targeted nanoparticulate carriers for delivery by noninvasive routes.

Keywords: Amnis image streamX Mk II imaging flow cytometer; Osteoporosis; Porcine sublingual mucosa; Quantification of intact NPs; Salmon calcitonin hydroxyapatite nanoparticles (SCT-HAP-NPs).